We have previously characterized intracisternal A-particle (IAP) genes as genetically distinctive retrovirus-like elements that are extensively reiterated in the cellular DNA of Mus musculus and some other rodent species. IAPs are not known to have an infectious extracellular phase. Last year we reported that IAP genes have typical retroviral long terminal repeat units (LTRs) and can act as mobile elements in the mouse genome. Cloned IAP LTRs were shown to promote CAT gene expression when introduced into the appropriate expression vector and transfected into either mouse or monkey cells. We have now found that LTR promoter activity is abolished or greatly reduced by specific methylation of HhaI or HpaII sites on either side of the RNA initiation site, an observation consistent with indirect evidence from this and other laboratories linking DNA mythylation with IAP gene expression in intact cells. IAP-specific sequences were found to be 5-10 fold enriched in polyadenylated RNA (polyA-RNA) from BALB/c thumus as compared to the polyA-RNAs from liver, spleen and kidney; IAP sequences were about 1/15th as concentrated in thymus as in several IAP-rich mouse tumors. Major IAP transcripts in BALB/c thymus were 7.2Kb and 5.4Kb in size and corresponded to IAP-associated RNA species previously extracted from mouse neuroblastoma cells. The amounts and relative proportions of these two transcripts varied in the thymuses of different inbred mouse strains, indicating that IAP expression in this tissue is under genetic control. In studies of BALB/c thymus DNA at the genomic level, the number of IAP 5 feet LTRs demethylated at the HpaII site was below the sensitivity of our detection method; i.e., very few of the 1000 IAP genes were actively transcribed. In situ hybridization of mouse and Syrian hamster (800 IAP elements per haploid genome) chromosomes showed multiple copies distributed over each chromosome. However, in the hamster, 50% of the IAP sequence was concentrated in blocks of constitutive, late replicating, non-centromeric heterochromatin. Clearly defined non-centromeric herochromatin is not found in the mouse. However, it is possible that in this species also, a large proportion of the IAP elements are sequestered in dispersed genetically silent regions of the chromosomes.